1 (1)

1. Introduction

2. Ocular drug delivery

3. Chitosan: why we prefer?

4. Chitosan-based drug delivery

systems for ocular application

5. Expert opinion

Review

Ocular application of chitosanEbru Basaran & Yasemin YazanAnadolu University, Faculty of Pharmacy, Department of Pharmaceutical Technology, Eskisehir,

Turkiye

Introduction: A major problem in ocular therapeutics with classical formula-

tions is the maintenance of an effective drug concentration at the site of

action for a long period of time. Enhancement of ocular bioavailability with

increased dose penetration and longer retention time at desired sites can

be achieved by recent formulations. Chitosan stands out with its unique

structural advantageous characteristics for different types of formulations

like in situ gelling systems, micro- and nanoparticles, inserts, etc.

Areas covered: In this review, the authors focus on ocular therapeutics and

the characteristics that make chitosan more acceptable in ocular applications.

Expert opinion: Chitosan seems to be one of the most promising polymeric

carriers for both hydrophilic and lipophilic drugs for ocular application.

Keywords: bioavailability, chitosan, ocular drug delivery, safety of chitosan

Expert Opin. Drug Deliv. (2012) 9(6):701-712

1. Introduction

Ocular drug delivery remains as one of the most challenging tasks for pharmaceuticalscientists with the unique pharmacodynamic and pharmacokinetic properties ofeyes [1,2]. The unique structure of the eye restricts the entry of drug molecules tothe required site of action. Therefore, the major problem in ocular therapeutics isto maintain an effective drug concentration at the site of action for an appropriateperiod of time in order to achieve the expected pharmacological response [3,4].Current ocular therapeutic options are unfortunately limited due to the low

systemic access owing to the blood--retinal, blood--aqueous and blood--vitreousbarriers. Oral therapy for ocular diseases requires high doses of active agent to reachtherapeutic concentrations at the site of action which may cause severe sideeffects [1,5]. The most common and well-accepted route is the topical administrationhaving two different purposes, to treat superficial diseases such as infections (e.g.,conjunctivitis, blepharitis, keratitis sicca) and to provide intraocular treatmentthrough the cornea for diseases such as glaucoma or uveitis [3,6-8]. Following topicalinstillation of an eye drop, drug is subject to a number of very efficient eliminationmechanisms such as tear drainage, binding to proteins, normal tear turnover,induced tear production and non-productive absorption via the conjunctiva. Typi-cally, drug absorption is virtually complete in 90 s due to the rapid removal ofdrug from the precorneal area. Additionally, cornea is poorly permeable to bothhydrophilic and hydrophobic compounds resulting in approximately 10% or lessabsorption of the topically applied dose into the anterior segment of the eye [9-12].Classical attempts for improving the ocular bioavailability of drugs mostly

include the use of viscosity enhancers (e.g., cellulose derivatives), mucoadhesivepolymers (e.g., polysaccharides) and in situ gel-forming systems [4]. As a conse-quence, many strategies were developed to enhance the bioavailability of drugsinstead of the standard treatment consisting of frequent instillations which cancreate incompliance, toxic side effects and cellular damage at the ocularsurface [12-14]. Among these approaches, two main strategies are to increase thecorneal permeability and to prolong the contact time with the ocular surface [7,15].

10.1517/17425247.2012.681775 2012 Informa UK, Ltd. ISSN 1742-5247 701All rights reserved: reproduction in whole or in part not permitted

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Penetration enhancers were used in many studies in orderto enhance the corneal penetration [4,6,16,17]. However, theseenhancers generally exhibit their effects by inducing morpho-logical changes in the corneal membrane and occasionally leadto adverse effects such as irritation in large doses. Therefore,the amount of penetration enhancers needs to be minimizedto prevent undesirable side effects [6,9].A further approach to optimize the efficacy of ocular dosage

form is the implementation of the mucoadhesive conceptwhich is successful in buccal and oral applications [13,18].Ocular bioavailability from a mucoadhesive dosage formdepends on the polymers bioadhesion properties which areaffected by its swelling, hydration time, molecular weightand degree of crosslinking. Other factors such as pH, mucinturnover and disease state also affect bioadhesion [19].Bioadhesion can be described in simple terms as the attach-

ment of a synthetic or biological macromolecule to a biologicaltissue. An adhesive bond may form either with the epithelialcell layer, the continuous mucus layer or a combination ofthe two. The term mucoadhesion is used specifically whenthe bond involves mucous coating and an adhesive polymericdevice while cytoadhesion is the cell-specific bioadhesion.The mechanism of bioadhesion between mucin and mucoad-hesive polymer is usually analyzed based on the molecularattraction and repulsion forces and depends on electronic,

adsorption, wetting, diffusion or fracture theories [20]. Dueto the attraction theories mentioned above, most cationicmacromolecules can interact with the anionic components ofsuperficial cellular glycoproteins. Moreover, the interior of tightjunctions (pores) is highly hydrated and contains constantnegative charges. A change in the relative concentration of spe-cific ion species in the pores causes alterations in tight junctionresistance leading to loosening or opening of the pores [12]. Themucus layer which is secreted onto the eye surface by the gobletcells is associated intimately with the glycocalyx of the corneal/conjunctival epithelial cells [13]. To prolong the residence timeof drugs in the preocular area, bioadhesive drug deliverysystems were developed taking advantage of the presence ofa mucin--glycocalyx domain in the external portion of theeye [20].Mucin is negatively charged at physiological pH (pH 7.4)

owing to the presence of sialic acid groups at the terminalends of the mucopolysaccharide chain resulting in the prefer-ential uptake of cationic drug carriers [19]. Therefore, use ofpositively charged formulations is the most commonapproach to enhance the bioavailability of the topicallyapplied ocular formulations. Development of molecularattraction between the negatively charged corneal and con-junctival surfaces with the positively charged formulationsby electrostatic interactions increases the bioavailability atthe ocular target site [21-24].Most common approach for designing a cationic formula-

tion is the addition of a cationic agent such as stearylamine(octadecylamine). This may lead to irritation and/or toxic effectand therefore more precaution is required at the configurationstep of the formulations [1,10,22,25]. Polymers with self-cationiccharacter can be used instead of adding cationic agents [26-28].The intimate contact ability of cationic mucoadhesive poly-meric systems will undoubtedly improve ocular bioavailabilityby high drug concentration at the absorbing corneal area result-ing in high drug flux through the absorbing tissue [4,29]. Pro-longed contact time may also increase the local permeabilityof high molecular weight drugs [9].Chitosan with its hydrophilic and cationic character is inves-

tigated widely for its potential as an excipient in oral andother pharmaceutical formulations. This polysaccharide isbeing studied for topical application for ophthalmic [4,9] andalso cosmetic purposes [30,31]. Chitosan has attracted a lot ofattention in ocular applications as a potential penetrationenhancer across the mucosal epithelia due to its polycationic,biocompatible and biodegradable nature together with itsmucoadhesive property [3,4,21,31,32]. Chitosan is generallyregarded as a non-toxic and non-irritant material and the pro-perties attributed to chitosan make it an excellent candidatefor ocular application [30].

2. Ocular drug delivery

The front part of the eye globe is clear and colorless and iscalled the cornea. It contains no blood vessels but is rich in

Corneal stroma

Bowmans membrane

Anterior corneal epitheliumstratified squamous

Keratocyte nuclei

Descemets membrane

Posterior endothelium

Figure 1. Layers of cornea.Reproduced with permission from Lutz Slomianka [81].

E. Basaran & Y. Yazan

702 Expert Opin. Drug Deliv. (2012) 9(6)

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nerve endings. Cornea consists of three major layers: outerepithelium, middle stroma and inner endothelium (Figure 1).When drug products are applied topically to the eye, they firstencounter the cornea and conjunctiva which represent theprimary barriers to drug penetration [33]. Drugs penetrateacross the corneal epithelium via the transcellular pathway(mainly for lipophilic drugs) or the paracellular pathway(for hydrophilic drugs). Transcorneal penetration seems tobe hindered by the binding of drug to the corneal tissueswhich are claimed to act as drug reservoirs [3]. The epitheliumand endothelium of the cornea are rich in lipid contentmaking them barriers to the permeation of polar, hydrosolu-ble compounds. The hydrophilic layer stroma contains70 -- 80% water and presents a barrier to the permeation ofnon-polar, liposoluble compounds [33]. Stroma, containingstructural and cellular elements including nerves, lymphaticsand blood vessels, is attached loosely to the underlyingsclera [34,35].Cornea shows permselectivity also. It has an isoelectric

point (pI) of 3.2. At pH values above the pI it carries a nega-tive charge and is selective for positively charged molecules.On the contrary, it carries a net positive charge at pH valuesbelow the pI. Conclusively, a positively charged moleculepasses through the cornea more effectively at physiologicalpH (7.4) than a negatively charged molecule [9].Conjunctiva is a thin transparent mucous epithelial barrier

which lines inside the eyelids and covers the anterior one-thirdof the eyeball (18 cm2) [34,36]. The respective portions of

conjunctiva are referred to as the palpebral and bulbarconjunctiva. Area joining palpebral and bulbar conjunctivais called the fornix (forniceal conjunctiva). Conjunctiva iscomposed of two layers, the outermost epithelium and theunderlying stroma (substantia propria). Epithelium is coveredwith microvilli and consists of stratified epithelial cells of5 -- 15 layers. Epithelial cells at the apical side connect witheach other by tight junctions which play a barrier role in per-meability [34]. Several polymers were identified that can safelyand reversibly disrupt cellular tight junctions. Among themchitosan appears to be a very promising candidate. In vitroand in vivo studies demonstrate chitosans ability to increasepassive diffusion of compounds across biological membranesprobably due to its effect on tight junction proteins [37].Pharmaceutical scientists show continuing interest in ocular

drug delivery due to challenges in unique pharmacodynamicand pharmacokinetic properties of the eye (Figure 2) [1,2].Selection and design of the route of administration of drugstake into account the quantity of drug present at the site ofaction to produce the desired action. Certain portions of theeye are more accessible to drugs given by one route than drugsgiven by another route [38]. For example, corneal route wasshown to be the predominant pathway for more lipophilic drugsfor delivery to iris while less lipophilic drugs need conjunctival/scleral penetration for delivery into the ciliary body [39].Drugs also vary in their ability to cross capillary and mucous

membrane barriers [38]. Common routes of administration foranterior-segment drug delivery are topical instillation and

Sclera

Rectus medialis

Hyaloid canal

Sulcus circularis corneaCiliary body

Zonular spaces

Choroid

Retina

Fovea centralis

A. Centralis retinaOptic nerve

Nerve sheath

Vitreous

Rectus lateralis

Posteriorchamber

Sulcus circularis corneae

Conjunctiva

CorneaIris

Lens

Body

Figure 2. Anatomy of the eye.Adapted with permission from RPS Publishing [36].

Ocular application of chitosan

Expert Opin. Drug Deliv. (2012) 9(6) 703

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subconjunctival injection while common routes for posterior-segment drug delivery include periocular and intravitrealinjections, systemic dosing and topical application [3,38].Direct injection can either be periocular (subconjunctival

or retrobulbar) or intravitreal. This type of application hasvery low patient compliance. Therefore, direct injection isused when relatively large doses of drug are required at thesite of action for immediate efficacy such as antibiotics andlocal anesthetics [38].When a drug is administered through the systemic route,

some portions of the eyes show no bioavailability because thoseportions are not vascularized (e.g., cornea) [38]. The seconddrawback of the systemic application is the exposure of thewhole organs to high doses of drug to maintain therapeuticconcentrations at the site of action which may result in severeside effects [1,5]. However, systemic drug administration maybe necessary for some instances to back up other treatmentslike the use of diuretics for treating glaucoma and as asupplementary to topical antibiotic application [38].Topical route is the most common route of administration

of drugs targeting both segments of the eye [3,38]. Topicallyapplied ocular agents produce effective levels mainly in theanterior segment [38]. There is high patient compliance over90% for topical administration because of the ease in useand no requirement for a qualified person for application(compared with ocular injections) [3,39].

3. Chitosan: why we prefer?

3.1 Source, structure and physicochemical properties

of chitosanChitosan is denominated to deacetylated chitins in variousstages of deacetylation and depolymerization and thereforenot easily defined in terms of its exact chemical composition(Figure 3) [30,40].

Chitin is present in the exoskeletons of crustaceans, cuticles ofinsects and cell walls of most fungi [41]. However, applications ofchitin are limited when compared with chitosan since chitin isstructurally similar to cellulose, but chemically inert. Acetamidegroup of chitin can be converted into an amino group to givechitosan by treating chitin with a concentrated alkali solution [42].Chitosan is a heteropolymer containing both glucosamine

and acetylglucosamine units [41,43]. Chitosan is one of themost promising polymers for drug delivery through themucosal routes because of its polycationic, biocompatibleand biodegradable nature as well as its mucoadhesive andpermeation-enhancing properties [3,21,31,32].Presence of amine group explains its unique properties

among other biopolymers, its cationic behavior in acidic solu-tions and its affinity for metal ions. Cationic metal sorptioncan occur either through chelation mechanisms in nearly neu-tral solutions or through electrostatic attraction; ion exchangefor metal anions happens to occur in acidic solutions [41].A clear nomenclature with respect to different degrees of

N-deacetylation between chitin and chitosan was not defined.Chitosan is not considered as one chemical entity but varies incomposition depending on the manufacturer [30].Commercially available chitosan is either in free base form or

in different salt forms. Hydrochloride, glutamate and lactatesalts being the most common, they appear as solutions, dryflakes and fine powders which are odorless, white or creamy-white, which vary in molecular weight in a wide range andvary also in degree of deacetylation and viscosity [30,44,45].Better mucoadhesion was observed for chitosan having

higher molecular weight (approximately 1400 kDa) comparedwith lower molecular weight chitosans (500 -- 800 kDa). How-ever, glutamate salt of a relatively lowmolecular weight chitosan(25 -- 50 kDa) also exhibited good mucoadhesion [46].Glass transition temperature of chitosan is 203C [13] and

chitosan degrades before melting which is a typical behaviorfor polysaccharides with extensive hydrogen bonding. Thisproperty makes it necessary to dissolve chitin and chitosanin appropriate solvent systems to impart functionality [45].However, fiber formation is quite common during precipita-tion and chitosan may look cottonlike. Aqueous solution(1% w/v) with the density of 1.35 -- 1.40 g/cm3 has a pHvalue of 4.0 -- 6.0 [30]. Chitosan exhibits both pseudoplasticand viscoelastic rheological behaviors [13].Chitosan does not cause allergic reactions or rejection due

to its biocompatible character. It breaks down slowly to harm-less amino sugars which can be absorbed completely by thehuman body [42].Various sterilization methods such as ionizing radiation,

heat, steam and chemical methods can be suitably adoptedfor sterilization of chitosan in clinical applications [42].

3.2 Ocular application of chitosan systemsChitosan is proposed as a material for potential ocular drug deli-very by reports claiming its biodegradability, biocompatibilityand good stability [47].

H H

Chitin

H

H

HH

H

NH

NH

H

HO

O

O

OO

C

CH3C

H3C

CH2OH

CH2OHHO

HO

O

H H

Chitosan

H

H

HH

H

NH2

NH2H

HO O

OO

CH2OH

CH2OHHO

HO

Figure 3. Structure of chitin and chitosan.Reproduced with permission from Elsevier [40].

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Approaches to penetration enhancement of topically applieddrugs considered chitosan as a superior mucoadhesive cationicpolymer due to its ability to develop molecular attraction forceswith the negative charges of mucin by electrostatic interactions.Interactions are determined by the formation of eitherhydrogen bonds or ionic interactions between the positivelycharged amino groups of chitosan and negatively charged sialicacid residues of mucin depending on the environmentalpH [13,48-50].Various chitosan derivatives were synthesized not only to

improve mucoadhesion but also to enhance penetration ofdrugs and peptides through the mucosa by opening the tightjunctions between epithelial cells or by intracellular routes.However, in vitro studies showed that cell surface bindingand absorption-enhancing effects were reduced in cell linescovered with mucus. Quaternized N-trimethyl chitosan andN-carboxymethyl chitosan were proved to be potent intestinalpenetration enhancers and therefore it was thought that theycould be of interest in ocular formulations when high aqueoushumor levels are required [13].Chitosan formulation needs no addition of organic solvent

during preparation since chitosan is soluble in weak acid sol-utions. Chitosan binds strongly to negatively charged cellularand mucosal surfaces resulting in the improvement of drugbioavailability and thus reduces the administration frequencywhich are all advantageous for controlled delivery [51].Many researchers studied chitosan formulations in the form

of several delivery systems like solutions, gels, liposomes, emul-sions, nanoemulsions, nanostructured lipid carriers (NLC),micro- and nanoparticles, inserts and chitosan conjugates aimingthe enhancement in the bioavailability of active agents [3,4,13].Felt et al. [14] studied the precorneal retention of tobramycin

in chitosan solutions and they found that even chitosan concen-tration as low as 0.5% is sufficient to ensure a significantenhancement in the residence time of ophthalmic solutions.Yuan et al. [11] demonstrated the feasibility of amphiphilic

chitosan self-aggregated nanoparticles as hydrophobic drugcarriers for ocular application with elevated retentionability at the procorneal area and no radioactivity in theposterior segment with a sustained release of entrapped drugover 48 h.De Campos et al. [21] showed the advantages of cationic sys-

tems in ocular drug delivery owing to their close contact withthe corneal and conjunctival surfaces. In this study, increase indelivery to external ocular tissues without compromisinginner ocular structures and providing long-term drug levelsin target layers could be maintained.Antibacterial activity of chitosan itself is also an advantage in

ocular application because secondary infections due to thediminished tear secretion containing antibacterial lysozymeand lactoferrin are frequently observed especially in keratoconjunctivitis sicca [13].It was reported that a radiolabeled chitosan formulation

remained at a constant viscosity at the ocular surface five timeslonger than other polymeric solutions [13].

Among the mucoadhesive polymers investigated, thecationic polymer chitosan has attracted a great deal of attentionbecause of its unique properties [21].

3.3 Polymer safetyChitosan was designated as Generally Recognized As Safe(GRAS) material in the USA not only as a pharmaceuticalexcipient but also for use in foods. It was also listed as afood additive in Japan, Italy and Finland [43,52,53].Chitosan has an industrial use as a flocculant and chelating

agent in the clarification of beverages and as a fungicide forcrop protection [43].Besides its food supplementary role, chitosan is being

investigated widely for use in pharmaceutical formulationsowing to its properties such as biodegradability, low toxicityand good biocompatibility [30,32,42,44,54].Levels of 4.5 -- 6.75 g chitosan taken daily by human

volunteers were shown to have no adverse effects. No clinicallysignificant symptoms including allergic response were found inshort-term human clinical trials of up to 12 weeks. However, alow incidence of mild and transitory nausea and constipationwas reported [43].Chitosan degrades under the action of ferments. Degradation

products are non-toxic and easily removable from the organismwithout causing concurrent side reactions. Chitosan possessesan antimicrobial property and absorbs toxic metals likemercury, cadmium, lead, etc. In addition, it has a good adhe-sion and coagulation ability and also an immunostimulatingactivity [42].Toxicity of chitosan was discussed in detail by Baldrick [43].

He concluded that the 50% lethal dose (LD50) of 16 g/kg (forrats 30 g/kg) body weight obtained in mice was close to sugaror salt and therefore chitosan can be classified as safe forpharmaceutical applications [42-44].Toxicities of chitosan and its derivatives evaluated by 50%

cellular growth inhibition (IC50) values against MCF7 andCOS7 cells were presented in the literature [55]. The resultingfinding was that most chitosans and their derivatives werenot significantly toxic compared with a toxic polymer suchas polyethylenimine (LD50 < 20 g/ml) [54].Yoksan and Chirachanchai [56] stated that LD50 of amphi-

philic chitosan nanospheres in rats are higher than 2 g/kgbody weight implying that the nanospheres are non-toxic.However, Omara et al. [57] reported alterations in liver enzymeseven at low doses of oral chitosan treatment in both male andfemale mice. Chitosan-induced histopathological changes(hypercellularity of glomerulus and degeneration in renaltubules with interstitial hemorrhage) were detected in liversand kidneys of rats. Increase in severity was found to bedose-dependent. Urea was significantly elevated mainly dueto hepatic failure caused by metabolic interruption inprotein metabolism which converts amino acids and ammoniato urea [57].The cytotoxicity and mucoadhesion behaviors of chitosan

nanoparticles were analyzed using lactate dehydrogenase and

Ocular application of chitosan

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MTS cell proliferation tests which are utilized to evaluate theeffect of nanoparticles on both cellular viability and mem-brane integrity [29]. Analysis results by Students t-test demon-strated no statistical difference between control media andnanoparticles with different sizes for the two tests. This resulthighlighted that chitosan nanoparticles are not harmful to thecells and the size has no effect on cytotoxicity [29].In parallel to researchers reporting no apparent toxic effects

observed for chitosan nanoparticles at low concentrations,death and malformation of zebrafish embryo occurred withincreasing chitosan nanoparticle concentration. Almost 100%of mortality was observed at a concentration of 40 mg/l for200 nm chitosan nanoparticles. Therefore, toxicity was decidedto be dose-dependent and considered at high concentrations. Itwas also found that small chitosan particles showed high toxi-city in the zebrafish embryo model identifying that toxicity ofchitosan nanoparticles was size-dependent [47].Loh et al. [58] studied the uptake and cytotoxicity of chitosan

nanoparticles in human liver cells and they concluded thatchitosan nanoparticles were less cytoadhesive than the chitosanmolecule itself. However, nanoparticles were rapidly interna-lized by bi-potential human liver cells (BHAL), the human livercell line derived from non-tumorous tissues. Internalized nano-particles were found to lead to a reduction in cell viability andproliferation while the extracellularly associated chitosanmolecules appeared to promote cell proliferation. Hence drugdelivery strategies using chitosan nanoparticles as a vehicleneed to consider their adverse effect on cells which are ofteninduced to proliferate in chronic liver disease [58].There are many examples of studies reporting the decrease

in chitosan toxicity following polyethylene glycolation(PEGylation) [45,59]. PEGylated chitosan was found to benon-toxic against cancer cells by Casettari et al. [45] and theyidentified the PEGylated chitosan copolymer as a promisingcandidate compound with potential use in a wide range ofbiomedical applications. IC50 of mitomycin-C decreasedfrom 1.97 0.2 to 0.13 0.02 g/ml when it was loadedinto chitosan oligosaccharide micelles and the IC50 value ofthe drug had no significant change when chitosan oligosac-charide micelles were PEGylated [60]. In their previous studieson Caco-2 cells, Prego et al. [59] reported that toxicity ofchitosan depends not only on its physicochemical propertiesbut mainly on the concentration of the polymer exposed toepithelium. Additionally, they also found that chitosan nano-capsules have a dose-dependent cytotoxicity, 50% LD50 beingaround 1 mg/ml. On the contrary, current analysis resultsshowed that PEGylated chitosan nanocapsules have a verygood biocompatibility with the monolayers. More specifi-cally, LD50 of chitosan--PEG nanocapsules was determinedto be between 10 and 20 mg/ml. This indicates that the cyto-toxicity inherent to chitosan nanocapsules was 10 -- 20 timesreduced owing to the PEGylation of chitosan [59].Parveen and Sahoo [61] studied chitosan/PEG blended poly

(lactic-co-glycolic acid) (PLGA) nanoparticles for tumortargeting drug delivery and they stated that PEG-modified

nanoparticles had the lowest percentage of uptake by macro-phages when compared with positively and highly negativelycharged nanoparticles. Both in vitro and in vivo resultsshowed that a combinatorial coating of PEG and chitosanled to a dramatic prolongation in blood circulation as wellas reduced macrophage uptake with only a small amount ofthe nanoparticles sequestered in the liver. It was further estab-lished that the surface properties affect the cytotoxicity profileof the nanoparticles. PEG and chitosan coating significantlyenhanced their cellular uptake and cytotoxicity in variouscancer cell lines. Based on the results mentioned above, thesestealth polymeric nanoparticles conferred by a combinatorialcoating of PEG and chitosan may suffice for long circulationserving as an efficient targeted drug delivery system [61].In conclusion, chitosan seems to have a potential for being

safe for ocular applications [43].

4. Chitosan-based drug delivery systems forocular application

Ocular application of chitosan formulations was studied bymany researchers. Among the formulations are solutions [14,62],gels [5,15,18,63,64], liposomes [10], emulsions [39], nanoemul-sions [25], NLC [7,23,65], micro- and nanoparticles [21,24,42,49,66,67],inserts [68] and chitosan conjugates [69].

4.1 Chitosan solutionsIt is clear that increase in both the retention time in precor-neal area and penetration of drug through the cornea are ofgreat benefit in ophthalmic therapy [48]. A significant increasein the corneal residence time of tobramycin was obtained withchitosan solutions when compared with the commercial drugsolution not only because of its ability to increase solutionviscosity but also because of its mucoadhesive properties [14].Topical application of chitosan solution has a penetration-

enhancing effect as expected from polycationic chitosan deriva-tives soluble at physiological pH of the tear fluid [37,62]. Thishypothesis was confirmed by preliminary results obtainedwith quaternary ammonium derivative N-trimethyl chitosanwhile N-carboxymethyl chitosan which is a polyanion at thephysiological pH of tear fluid failed to enhance intraoculardrug penetration significantly [62].Efforts were oriented toward the addition of polyol salts

bearing a single anionic head such as glycerol-, sorbitol-,fructose- or glucose-phosphate salts (polyol- or sugar-phosphate)to chitosan aqueous solutions. Proposed salts are ideal agentsfor transforming pH-dependent chitosan solutions intotemperature-controlled pH-dependent chitosan solutions.Combination of chitosan, a cationic polysaccharide, andpolyol-phosphate salt benefit from several synergistic forcesfavorable to gel formation including hydrogen bonding, electro-static and hydrophobic interactions. Phosphate salts givechitosan a unique behavior by allowing chitosan solutions toremain liquid at physiological pH and turn into gel if heated atbody temperature. Uniqueness of chitosan solution resides also

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in overcoming pH barrier which has long been a major limi-tation for many applications. Thus, the phenomenondescribed here is quite distinct from that shown by solutions ofwater-soluble cellulose modified hydrophobically by ionicsurfactants [70].Gratieri et al. [5] investigated the ocular delivery of fluco-

nazole by topical application of chitosan solution and polox-amer/chitosan in situ gel-forming formulation. The studydemonstrated that poloxamer/chitosan in situ gel-formingformulation retarded fluconazole release when compared withthe chitosan solution. However, chitosan solution was able todeliver higher fluconazole amounts than in situ gel-formingformulation in ex vivo studies on porcine cornea. Both formu-lations exhibited similar sustained release behavior in thein vivo study. It was concluded that both chitosan solutionand in situ gelling formulation have the potential of treatingfungal keratitis.Solutions outstand with their simplicity in preparing and

constitution with commercially available polymers at relativelylow costs [5].

4.2 In situ gelling systemsIn situ gelling systems can be easily and accurately applied inliquid form to the surface of the eye where crosslinkage occurswith the cations present in tear fluid in order to form a gel onthe ocular surface. This is thought to prolong the precornealretention time and thus lead to increased bioavailability ofthe drug [15].Rapid turnover rate of the lacrimal fluid generally leads to a

dilution of common viscous eye drops. Enhancement of the vis-cosity of formulations can be detected depending on theincrease in the amount of cations present [15]. In situ gellingmechanisms can be triggered either by pH or by temperaturechanges [18,63,64,71]. Chitosan-based hydrogels can be formu-lated by the addition of a crosslinker to form covalent or ionicinteraction between polymeric chains. Hydrogels can also beformed by complexation with another polymer generally byionic interaction or by aggregation after chitosan grafting [72,73].Covalently crosslinked hydrogels are the only systems characte-rized by a permanent network due to their irreversible chemicallinks. Therefore, they exhibit good mechanical properties andcan overcome dissolution even in extreme pH conditions. Onthe other hand, other types of hydrogels are more labile.Covalent crosslinkers necessary to obtain hydrogels are eitherknown to be relatively toxic or their fate in the human body isunknown and/or there is a lack of data concerning their bio-compatibility. Therefore, an additional purification and verifi-cation step is required before administration of the hydrogelsince it may be problematic if free unreacted crosslinker is foundin traces before administration [72].Gratieri et al. [18] prepared an in situ gel-forming formula-

tion by combining poloxamer/chitosan and they obtainedimproved mechanical and mucoadhesive properties as wellas enhanced retention time on mucin discs. Gels preparedproved to possess a mucoadhesive ability which is influenced

by chitosan concentration. Gamma scintigraphic analyses inhumans confirmed the prolonged retention time of the for-mulation due to faster gelling of the chitosan formulationunder in vivo conditions which makes drainage more difficult.Delivery system developed seems to be a promising tool forophthalmic use because it is easily administered and shows aprolonged ocular contact time [18].Sustained ophthalmic drug delivery of baicalin over 8 h was

obtained by pH-triggered in situ gelling system [74]. Formula-tion caused no irritation when tested on rabbit eye tissues.Both in vitro and in vivo results indicated that the pH-triggeredin situ gelling system was a viable alternative to conventionaleye drops by virtue of its ability to enhance bioavailabilitythrough longer precorneal residence time and ability to sustaindrug release. More importantly, gelling system can be consi-dered as a novel ophthalmic delivery system being a suitablemedium for pH-sensitive baicalin [74].Physicochemical and rheological properties of a novel

thermo-sensitive hydrogel system based on chitosan and inor-ganic phosphate was studied as a function of temperature [63].There are two phase transition points as a function of temper-ature which corresponds to 30 and 43C. While gel formationof the system at low temperature (< 30C) was reversible, gelformation at high temperature (> 43C) was irreversible. Refer-ring to the results on pH value, conductivity and ionic strengthchanges as a function of temperature, it seems that hydrogenbonding between chitosan skeleton and water molecule andalso the physical conjunctions may be the main driving forcesto gel formation at low temperature (< 30C). However, gelformation at high temperature (> 43C) may be resultingfrom hydrophobic interactions [63].

4.3 LiposomesUse of colloidal drug delivery systems such as liposomes is asuitable strategy to obtain enhanced ocular bioavailability incomparison with liquid formulations. Liposomes are preferredbecause they exhibit unique features by offering easy delivery,no interference with vision and stabilizing drug as an excellentreservoir [75]. On the other hand, liposomes are generally ratherunstable and tend to degrade or aggregate and fuse which leadsto the leakage of entrapped drug during storage or after admi-nistration. Among the many attempts aiming to minimize thedisruptive influences is surface modification of liposomes whichcan improve liposomal stability both in vitro and in vivo [75].It was reported that positively charged liposomes had a

higher binding affinity to the corneal surface than the neutraland negatively charged vesicles as a result of interactionbetween positively charged liposomes and polyanionic cornealand conjunctival surfaces. However, cationic lipids such asstearylamine incorporated to give positive charge to liposomesmay lead to irritation and toxic effects [10].Formation of a bioadhesive and polymeric membrane

around the liposomes was investigated [75]. Most of the selectedmembrane materials for liposomes were chitosan based [4,10].Li et al. [10] studied low molecular weight chitosan-coated

Ocular application of chitosan

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liposomes and their potential use in ocular drug delivery.Researchers found that the coating layer added positive chargeas well as an excellent bioadhesive property. Precorneal reten-tion was significantly prolonged by chitosan coating comparedwith either non-coated liposome or drug solution. Chitosancoating also demonstrated an improved transcorneal drug pen-etration rate which was attributed to the penetration-enhancingeffect of chitosan. Meanwhile, chitosan coating displayedpreferable physicochemical stability and pronounced in vivoocular tolerance [10].Rational mixture of chitosan with phospholipids or with

preformed liposomes led to formation of supramolecularhybrid structures [4]. These nanostructures demonstrated anadequate stability in biological fluids and are suitable for theencapsulation of labile macromolecules. They have an addi-tional property of ability to control the release of entrappedmolecules as a function of lipidic composition. It was shownthat chitosan--lipid nanocomplexes can interact efficientlywith ocular tissues and enter the ocular cells withoutcompromising the integrity of cellular membrane.Diebold et al. [76] analyzed the ocular application of

liposome--chitosan nanoparticle complexes. Results of thestudy showed that nanosystems consisting of chitosan andphospholipids are first retained in the mucus layer and thenenter the conjunctival cells at different levels depending onthe composition. Furthermore, these nanosystems exhibitednegligible toxicity in vitro and a good tolerance in vivopointing out that liposome-chitosan nanoparticle complexesare promising candidates for drug delivery through the ocularmucosa [76].

4.4 EmulsionsConventional ophthalmic dosage forms tend to be either simplesolutions of water soluble drugs or suspensions of waterinsoluble drugs. Unfortunately, these delivery systems generallyresult in poor corneal drug absorption and therefore most of thedrug applied does not reach the intended site of action. Micro-emulsions may offer a solution to the problem of poorcorneal delivery by sustaining the release of the drug andalso by providing a higher penetration into deeper layers ofthe eye. In addition, microemulsions have the potential ofincreasing the solubility of the drug in the vehicle [3].In 2002, the Food and Drug Administration (FDA)

approved the clinical use of an anionic emulsion containing0.05% cyclosporine A (Restasis, Allergan, Inc., Irvine, CA,USA) for the treatment of chronic dry eye. Either cationicor anionic nanoemulsions were recently approved for thetreatment of ocular inflammations and for other oculardisorders [4].Cationic emulsion was reported as being more effective in

increasing the uptake of drugs in various ocular tissues followingtopical administration when compared with solutions oranionic emulsions [25].Negatively charged emulsions can be prepared using anionic

lipids and surfactants while positively charged emulsions using

cationic lipids such as stearylamine and oleylamine. Alterna-tively, cationic polysaccharides such as chitosan can be usedto form a coating around the oily droplets thus impairing apositive charge to the emulsion [4]. Unfortunately, stearylamineshowed in vitro high toxicity against the tested cell systems [1,10].Cytolytic and cytotoxic activity limits the consideration of thesesystems as novel drug delivery carriers [77].Chitosan has proved to be a useful emulsifier that stabilizes

emulsions and prevents coalescence by steric and electrostatichindrance without the help of additional surfactant due to itsself-cationic character [4].Drugs incorporated into oil-in-water emulsion (o/w) are

lipophilic in nature and either corneal or conjunctival/scleraroute of penetration is favored depending on the extent oflipophilicity [39].

4.5 Particular systemsMicro- and nanoparticles were shown to be efficientocular delivery systems [22,27,28,49]. Since composition of thecolloidal system may affect its affinity to the ocular mucosa,several approaches were investigated for the ultimateformulation [22,28,48,49,66].Microspheres have the potential of being used for targeted

and controlled release drug delivery. Addition of bioadhesiveproperties to microspheres has additional advantages, forexample, efficient absorption and enhanced bioavailabilitydue to a much more intimate contact with the mucus layerby high surface to volume ratio, and specific targeting of drugsto the absorption site [20].Bioadhesive microspheres can be tailored to adhere to any

mucosal tissue thus offering the possibility of localized or sys-temic controlled release systems. Application of bioadhesivemicrospheres to the mucosal tissues of ocular cavity is usedfor administration of drugs mostly for local action [20].Prolonged release of drugs and a reduction in fre-

quency of ocular administration can highly improve patientcompliance [20].Two approaches regarding ocular application of chitosan

particles are incorporation of active agent into chitosannano- and microparticles or chitosan-coating of eitherpolymeric or lipidic particles [20,21,66].In a study by Yuan et al. [66], ocular application of rapamycin-

incorporated chitosan nanoparticles and rapamycin suspensionwas compared. Ocular distribution results showed that bothformulations showed good spreading characteristics over theentire precorneal area just after topical administration. Animalstreated with rapamycin-incorporated chitosan nanoparticlespresented significantly higher (p < 0.05) remaining radioacti-vities on corneal and conjunctival surfaces than those treatedwith rapamycin suspension (two to six times increase; for atleast 24 h). Enhanced duration on ocular surfaces was attributedto the mucoadhesive character of chitosan mediated by theelectrostatic interaction between positively charged chitosanand negatively charged corneal and conjunctival cells. Theyconcluded that chitosan formulations can improve the residence

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time of drug on tissues and cells, release the drug in a sustainedpattern and thus improve the bioavailability of drug and reducethe administration frequency [11,51,66].De Campos et al. [21] investigated the potential use of chito-

san and chitosan-coated nanoparticles for specific delivery ofdrugs to the ocular mucosa. They considered the advantagesof intimate contact tendency with the corneal and conjunctivalsurfaces, increasing delivery to only external ocular tissues andmaintaining long-term drug levels. Systems showed greatpromise with at least 24 h corneal and conjunctival residencetime [21,49].Mucoadhesive chitosan--sodium alginate nanoparticles

were investigated as a new vehicle for prolonged ophthalmicdelivery of an antibiotic, gatifloxacin [67]. Analyses resultsshowed that the drug was released from the optimized formu-lation over a period of 24 h in a sustained release mannerprimarily by non-Fickian diffusion. Formulation preparedwas proposed to be a viable alternative to conventional eyedrops by virtue of its ability to sustain drug release, ease inadministration and reduced dosing frequency resulting inbetter patient compliance.The second generation of submicron particles, NLC can also

be used as topical drug delivery system for ocular mucosa [65,78].NLC combines many features of pharmaceutics, that is,prolonged release of actives, drug targeting and increasingamount of drug penetrating into mucosa. NLC exhibits anexcellent tolerability due to the physiological and/or biode-gradable lipids used in the formulation [23]. Studies indicatedthat NLC increases the ocular bioavailability of lipophilic drugswithout inducing discomfort or irritation [7,79]. Resultingfindings of prolonged precorneal residence time and deliveryto ocular surface and anterior chamber showed that thiolatedNLC is a promising strategy to the treatment of ocular surfaceand anterior segment inflammatory diseases (e.g., uveitis) [65].When the potential of chitosan-coated NLC was investigated

for ocular delivery, it was found that positive charge of NLCdispersions provided a longer retention time by interactingwith the negatively charged mucous. Eventually, an improvedpenetration rate was achieved by the presence of chitosan con-cerning its effective contribution to the corneal permeability.The most notable advantage of chitosan-coated NLC was theirsuperior mucoadhesive properties [23].Existence of the bioadhesive polymer chitosan on nanocap-

sules was concluded to provide an optimal corneal penetrationof encapsulated drugs with good ocular tolerance. Chitosan-coated colloidal drug carriers were proposed as promisingsystems to overcome the present limitations in ocular drugdelivery [23,24,48].

4.6 Other delivery systemsFilms, erodible and non-erodible inserts, rods and shields arethe most logical delivery systems aiming the long remainingtime on ocular surface. These delivery systems sustain andcontrol drug release and thus avoid pulsed entry characterizedby a transient overdose, followed by a relative short period of

acceptable dosing which in turn is followed by a prolongedperiod of low dosing [13].Mono- and bilayer dexamethasone-chitosan films were

successfully obtained and their release tests suggested that thefilms are potential sustained-release carriers for dexamethasone.Incorporation of a second layer of chitosan film modified drugrelease profile significantly. As a conclusion, while the mono-layer dexamethasone-chitosan film is promising for dexametha-sone for a few hours, bilayer dexamethasone-chitosan filmseems to be promising for weeks [80].Di Colo et al. [68] prepared an insert aiming enhanced

ocular bioavailability of ofloxacin. Following insertion, everyinsert formed a superficial gel, adhered to the applicationsite and then gradually spread over the cornea and eroded.While remarkable bioavailability increase was determinedcompared with commercial eye drop, signs of mild irritationwere seen [68].

5. Expert opinion

Topical ocular route of administration is preferred for manydrugs due to ease in access and high patient compliance whentreating diseases at both anterior and posterior segments. Pro-viding a sufficient dose at the desired site of action is a greatchallenge for ocular therapeutics due to anatomic and physio-logic barriers of the eye limiting drug delivery especially to theposterior segment tissues. Most common approaches for theenhancement of ocular bioavailability are prolonging retentiontime and enhancement of ocular penetration. Cationic lipidsused widely for those purposes were limited by their cytotoxiceffects. As an alternative, self-cationic polymers like chitosangained more attention in ocular applications. Several typesand derivatives of chitosan can be tailored from chitin leadingto a possibility of selecting the most appropriate chitosan typeto obtain the desired characteristics of delivery systems forboth hydrophilic and lipophilic drugs.Aiming enhanced ocular bioavailability, different drug

delivery systems are as important as the polymeric structure.Due to the transcendent properties of the polymer, manyformulations were developed using chitosan. Among those,particulate systems seem to be the most promising system forocular applications. Considering the superior characteristics ofparticulate systems like enhanced stability of the drug incorpo-rated, perdurable particulate structure, high drug payload,controlled release of the actives, etc., incorporation of chitosaninto those systems contributes to the properties mentioned.Emulsification/solvent evaporation, spray-drying, ionotropic

gelation and coacervation techniques using chitosan results innano/microparticulate systems while avoiding organic solventsand preventing coalescence caused by steric and electrostaticinteractions owing to chitosans emulsifying character, possi-bility of incorporating both hydrophilic and lipophilic drugswhich makes chitosan more preferential.PEGylation must be taken into account when safer and

more effective particulate formulations are required. Either

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chitosan-based or chitosan-coated particles can be PEGylatedresulting in improvement of in vitro--in vivo stability, decreasein toxicity and enhancement of ocular penetration due toenhanced mucoadhesive characteristics of the nanocarriers.Chitosan itself also enhances the penetration of drugs by

opening the tight junctions between epithelial cells or byintracellular routes. Chitosan provides the drug to enter theocular cells without disturbing the integrity of cellularmembrane and further PEGylation leads to more enhancedmucoadhesion. Since nature of coating affects the interaction

with the epithelial cells and also transport across the cornealepithelium, precise concentrations are required.Overviewing the outstanding features of chitosan, it seems

to gain increasing attention in the treatment of severeocular disorders.

Declaration of interest

The authors state no conflict of interest and have received nopayment in preparation of this manuscript.

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AffiliationEbru Basaran1 & Yasemin Yazan2

Author for correspondence1Assistant Professor,

Anadolu University,

Faculty of Pharmacy,

Department of Pharmaceutical Technology,

26470 Eskisehir, Turkiye

Tel: +90 222 3350580 ext. 3739;

Fax: +90 222 3357170;

E-mail: [email protected],

Anadolu University,

Faculty of Pharmacy,

Department of Pharmaceutical Technology,

26470 Eskisehir, Turkiye

E. Basaran & Y. Yazan

712 Expert Opin. Drug Deliv. (2012) 9(6)

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